Cell-cell recognition studies are currently hindered by the lack of rapid biochemical assays for the recognition processes. This is particularly a problem in many important immunological systems where only a small fraction of the cell population responds, or where the kinetics of the response varies from cell to cell. A potential solution to this problem is to utilize fluorescent probes of intracellular function and to follow cellular responses with flow cytometry techniques. Several parameters that have been monitored in intact cells with optical techniques and are adaptable to flow cytometry analysis are: a) reduced pyridine nucleotide [NADP(P)H] levels, b) cellular light scattering properties, c) membrane-bound Ca++ levels, d) intracellular free Ca++ levels, e) transmembrane electrical potential, and f) intracellular pH. In the present studies, changes in the above parameters will be monitored during receptor-mediated triggering of the respiratory burst of O2-production in neutrophils and macrophages. Triggering by the soluble agonist 12-0-tetradecanoyl phorbol-13-acetate (TPA) and the chemotactic peptides F-Met-Leu-Phe and C5a will be studied and compared to triggering by immune target membranes bearing IgG or C3b. Studies to date of NAD(P)H levels in neutrophils have shown that TPA triggers the respiratory burst by a cooperative mechanism such that cells are quantized in either resting or activated states. One purpose of the proposed studies is to determine whether a similar cooperative process operates in triggering by the above physiological agonists. Because each of these agonists acts on a distinctly separate cell surface receptor, the results will indicate whether the cooperative process is unique to individual receptors, or whether it is a common underlying pathway that is coupled to receptor action. Analysis of the above intracellular biochemical parameters (a-f) should reveal both the biochemical changes that precede and sequentially follow the cooperative event. Furthermore, once measurements of the biochemical changes are successfully made during the activation process, it will be possible to resolve the temporal sequence of events by pairwise analysis of the individual changes using dual beam flow cytometry. Because of cell to cell variation in temporal response, this single cell technique promises to provide much higher time resolution than has been possible with methods which must average the response of many cells. An immediate clinical application of this work is a rapid screening test for chronic granulomatous disease and the genetic carrier state.